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Experiment 6A, 6B: Chromatography

Rachelle Anne Victorio
CHM 342.003
03 February 2016
Ms. Emily Babusa

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Purpose:
To give practice in separating components of a mixture using chromatography. To
determine which solvent would be best for separating a pair of related compounds that
differ slightly in polarity. To identify compound(s) present in an unknown sample.

Table of Chemicals:
Molecular
Formula

Structural Formula

Molecular
Weight

Boiling /
Melting

Density

Methylene
Chloride

CH2Cl2

84.93 g/mol

-96.7C /
39.6C

1.33 g/mL

Fluorene

C13H10

166.223 g/mol

295C/116
C

1.2 g/mL

Fluorenol

C13H10O

182.22 g/mol

367.511.0
C/152 to
155 C

1.151
g/mL

Fluorenone

C13H8O

180.192 g/mol

342C/
83.5oC

1.13 g/cm

Iodine
Crystals

I2

253.80894
g/mol

184C/114
C

11.27 g/L

Benzoin

C14H12O2

212.24 g/mol

344oC/132o
C

1.31 g/mL

(CHCO)

210.23 g/mol

346-348oC/
94-96oC

1.23
g/mol

C6H14

86.18 g/mol

-95.3 C /
68.7 C

0.66 g/mL

Benzil

Hexane

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Acetone

C3H6O

58.08 g/mol

56oC/-95oC

0.79 g/mL

Outlines of Procedure:
Experiment 6A: Thin Layer Chromatography
In this experiment, you will use thin-layer chromatography (TLC) to separate a mixture of three
compounds: fluorene, fluorenol, and fluorenone.

Based on the results with known samples of these compounds, you will determine which
compounds are found in an unknown sample. Using TLC to identify the components in a sample
is a common application of this technique.
Preparing the TLC:
Technique 20 describes the procedure used for thin-layer chromatography
1. Use a 10cm x 5.3 cm TLC plate (Whatman Silica Gel Plates No. 4410 222). These plates
have a flexible backing but should not be bent excessively. They should be handled carefully or
the absorbent may flake off. Also, they should be handled only by the edges; the surface should
not be touched.
2. Using a lead pencil (not a pen), lightly draw a
line across the plate (short dimension) about 1 cm
from the bottom (see figure.)
3. Using a centimeter ruler, move its index about
0.6 cm in from the edge of the plate and lightly
mark off five 1-cm intervals on the line. These are
the points at which the samples will be spotted.
4. Prepare five micro-pipettes to spot the plate.
*The preparation of these pipettes is described
and illustrated in Technique 20, section 20.4.
5. Prepare a TLC development chamber with
methylene chloride (see technique 20, figure 20.5). A beaker covered with aluminum foil or
wide-mouth screw cap bottle is suitable container to use. (see tec. 20, fig. 20.4). *The backing on
the TLC plates is to thin, so if it touches the filter paper liner of the development chamber at any

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point, solvent will begin to diffuse onto the absorbent surface of that point. To avoid this, be sure
that the filter paper liner does not go completely around the inside of the container. A space of
2.5 inches must be provided.
6. On the plate, starting from left to right, spot fluorene, fluorenol, and fluorenone, the
unknown mixture, and the standard reference mixture which contains all three compounds.
7. For each of the five samples, use a different micropipette to spot the sample on the plate. *The
correct method of spotting is described in Tec. 20, sec 20.4.
8. Take up part of the sample in the pipette (dont use a bulb, capillary action will draw up
liquid).
9. Apply the sample by touching the pipette lightly to the thin-layer plate. *The spot should not
be larger than 2mm in diameter. It will usually be sufficient to spot each sample once or twice. If
you need to spot the sample more than once, allow the solvent to evaporate completely between
successive applications and spot the plate in exactly the same position each time.
10. Save the sample in case you need the TLC.
Developing the TLC Plate:
1. Place the TLC plate in the development chamber, making sure that the plate does not come in
contact with the filter paper liner.
2. Remove the plate when the solvent front is 1-2 cm from the top of the plate.
3. Using a lead pencil, mark the position of the solvent front.
4. Set the plate on a piece of paper towel to dry.
5. When the plate is dry, place the plate in a jar containing a few iodine crystals (see tec. 20, sec,
20.7), cap the jar, and leave it in the car until the spots begin to appear.
6. Remove the plate from the jar and lightly outline all the spots that became visible with the
iodine treatment.
7. Using a ruler, marked in millimeters, measure the distance that each spot has traveled, relative
to the solvent front.
8. Calculate the RF values for each spot (tec 20, sec 20.9).
9. Explain the relative positions of the three compounds in terms of their polarities. Identify the
compound or compounds found in the unknown mixture.
10. With your instructors option, submit the TLC plate with your report.
Experiment 6B: Selecting the correct Solvent for Thin-Layer Chromatography
In experiment 6A, it was stated what the solvent to use for developing TLC plate. In some
Compounds: Benzoin and Benzil

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Solvents: Acetone, Methylene Chloride, Hexane

Preparation:
1. A pair of compounds will be assign to run on TLC, you selecting an own pair.
2. Obtain about 0.5 mL of the three solutions: one solution of each of the two individual
compounds and a solution containing both compounds.
3. Prepare three thin-layer plates in the same way in Experiment 5A, except each plate should be
10 x 3.3 cm.
4. When marking them with a pencil for spotting, make three marks 1 cm apart.
5. Prepare three micro-pipettes to spot the plates.
6. Prepare three TLC development chambers (like experiment 5A), with each chamber
containing one of the three solvents suggested for your pair of compounds.
Developing the TLC plate:
1. On each plate, spot the two individual compounds and the mixture of both compounds.
2. For each of the three samples, use a different micropipette to spot the sample on the plates.
3. Place each TLC plate in one of the three development chambers, making sure that the plate
does not come in contact with the filter paper liner.
4. Remove each plate when the solvent from is 1-2cm from the top of the plate.
5. Using a lead pencil, mark the position of the solvent front.
6. Set the plate on a piece of paper towel to dry.
7. When the plate is dry, observe it under a short-wavelength UV lamp, preferably in a darkened
hood or a darkened room.
8. With a pencil, lightly outlines any spots that appear.
9. Next, place the plate in a jar containing a few iodine crystals, and leave it in the jar until to
spots became visible with the iodine treatment.
10. Using a ruler, marked in millimeters, measure the distance that each spot has traveled relative
to the solvent from.
11. Calculate the Rf values for each spot. *With the instructors instruction, submit the TLC
plates with the report.
12. Keep in mind: Which of the three solvents resolved the two compounds successfully? For the
two solvents that did not work, explain, in terms of their polarities, why they failed.

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Observations:
The first part of the experiment in an introduction to the thin layer chromatography. TLC
was used to separate the three compounds: fluorene, fluorenol, and fluorenone. The experiment
started by preparing the development chamber which consists of approximately about 1-3mL of
methylene chloride inside the medium beaker, and a filter paper wrapped around the inside of the
beaker. The beaker chamber was then covered by a foil to avoid contamination. TLC paper was
prepared with the instructed measurement distances. Using a pipette, spotting was started from
left to right (fluorene, fluorenol, fluorenone, the unknown mixture, the mixture of three
compounds). The unknown that used in this experiment is, unknown 2. After spotting, the TLC
paper was placed inside the chamber, making sure that the plate wont come in contact with the
filter paper. Developing the TLC plate until the solvent (methylene chloride) reach the solvent
front, which is about 1-2cm from the top of the plate, takes about 10-15 minutes. At first, the
TLC plate was placed inside a jar with a few iodine crystals. Spots starting to appear after about
6-10 minutes. The plate was removed from the iodine jar and were tried to view the spots under
the UV light. More spots are visible under the UV light. The possible reason behind the spots not
very visible on the iodine crystal, is that it takes time for it to develop. The farthest spot is
fluorene (55 mm), then fluorenone (37 mm) and lastly fluorenol (19 mm). The unknown mixture
shows that its the combination of the three compounds (fluone, fluorenol, fluorenone).
The same process was used on the experiment 6B. However this time, benzoin and
benzyl was used, and the solvents are acetone, methylene chloride, and hexane. In this
experiment, the three solvents are tested to find out which one is the best TLC solvents will work
best on separating the compounds. During the experiment, observation shows that acetone
evaporated quickly and when the TLC plates are observed under the UV light, the compounds
moves on the same rate. This concludes that acetone isnt the best solvent. The TLC plate from
the solvent hexane was then observed under the UV light, it shows that the spots didnt move at
all, therefore its not the best solvent. The TLC plate from the methylene chloride shows the best
separation of the spots (compounds). Some spots were shown due to contamination. However,
the compounds spots are still visible. By observation, benzyl traveled the farthest on the plate
with a distance of 43 mm, then benzoin with a distance of 25 mm.

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Results and Discussion:

Part 6A: Fluorene, Fluorenol, and Fluorenone in Methylene Chloride
1)
The first image below shows the TLC paper under the UV light. Measurements of the distances
are obtained and recorded below:

Using the distances that were measured from the TLC paper, the Rf values are calculated and
shown on the table below:
Compound:

Rf Values:

Calculations:
Rf =
Distance
Distance
start center of substance spot start solvent front

(1) Fluorene
(2) Fluorenol
(3) Fluorenone
(4) Unknown 2
(Fluorene, Fluorenol,

1.0
0.35
0.67
1.00
0.35

(1)
(2)
(3)
(4)

=55 mm/55 mm
=19 mm/55 mm
= 37 mm/55 mm
=55 mm/55 mm
=19 mm/55 mm

=1.00
=0.35
=0.67
=1.00
=0.35

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Flurenone)
(5) Mixture
(Fluorene, Fluorenol,
Fluorenone)

0.67
1.00
0.35
0.67

(5)

= 37 mm/55 mm
=55 mm/55 mm
=19 mm/55 mm
= 37 mm/55 mm

=0.67
=1.00
=0.35
=0.67

(2)
In this thin layer chromatography experiment, the three compounds used were fluorene,
fluorenol, and fluorenone. Methylene chloride was used as a solvent for all of these. The Rf
values for fluorene is 1.00. Based on the structure of fluorene, which is a completely flat and
non-polar compound, and methylene chloride, a moderately polar molecule, the Rf value
calculated was accurate. This is because the methylene chlorise was too polar in comparison to
the fluorene to be an ideal solvent, so the fluorene transferred all the way to the top of the
chromatography paper.
The Rf values for fluorenol was 0.35. Since the fluorenol is a very non-polar molecule because of
the phenyl groups, and the methylene chloride has moderate polarity, it is predicted that the Rf
values for the fluorenol would be very low.
The Rf values for fluorenone was 0.67. This is expected because methylene chloride and
fluorenone have similar polarities, which allows the methylene chloride to move fluorenone
about halfway up the chromatography paper.
(3)
The unknown 2 are composed of the mixture of the three compounds (fluorene, fluorenol,
fluorenone). The Rf values that are calculated are: fluorene: 1.00, fluorenol: 0.35, fluorenone:
0.67. The Rf calculations for all compounds are just the same as the one calculated on individual
test. The same results are calculated on the mixture compounds.

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Part 6B:
(1)
Benzoin and Benzil are the compounds used in this experiment, and the solvents used are:
acetone, methylene chloride, and hexane.
The following plates are results from the experiment:

Benzoin, Benzil and

mixture in Acetone

Benzoin, Benzil and

mixture in Methylene
Chloride.

Benzoin, Benzil and

mixture in Hexane

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Using the TLC plates, the distances are measured and shown below:

(2)
The two compounds tested in this experiment part 6B were benzoin and benzil. The solvents
were tested in acetone, methylene chloride, and hexane. The first test was in acetone. As shown
on the pictures on the previous page, the compounds (benzoin, benzyl, mixture), all moved at the
same rate. The second test was in methylene chloride, as shown in the picture, the compounds
moved halfway up the paper plate, and separating on the last one (mixture) neatly. The last test is
in hexane, as shown in the picture, the spots (compounds) didnt move at all.
This experiment shows that the best solvent for benzoin and benzil for TLC is methylene
chloride.
The table below shows the Rf values for hexane:
Rf Values for Part B (in Methylene
Chloride)
Compound
Rf Values

Rf=

Distance
Distance
start center of substance spot start solvent front

Benzoin, Benzil and

mixture in Acetone

Benzoin

Calculations:

0.38

Benzoin, Benzil and

mixture in Methylene
Chloride.
=25 mm/66 mm

Benzoin, Benzil and

mixture in Hexane

=0.38

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Benzil
Mixture (Benzoin
and Benzil)

0.65
0.38, 0.65

=43 mm/66 mm
=25 mm/66 mm
=43 mm/66 mm

=0.65
=0.38
=0.65

By using the Rf values, observation shows that benzil traveled further than benzoin. Benzil
having a 0.65 Rf value and benzoin having a Rf value of 0.38.
(3)
The table below shows the Rf values for acetone.
Rf Values for Part B (in Acetone)
Compound
Rf Values

Calculations:
Rf=

Distance
Distance
start center of substance spot start solvent front

Benzoin
Benzil
Mixture (Benzoin
and Benzil)

0.72
0.72
0.72

=34 mm/ 47 mm
=34 mm/ 47 mm
=34 mm/ 47 mm

=0.72
=0.72
=0.72

Using acetone as the solvents shows that the compounds all moved at the same rate. The reason
behind this is that, acetone polarity to too high to effectively separate benzoin and benzil from
the TLC plate.

The table below shows the Rf values for Methylene Chloride:

Rf Values for Part B (in Hexane)
Compound
Rf Values

Calculations:
Rf=

Distance
Distance
start center of substance spot start solvent front

Benzoin
Benzil
Mixture (Benzoin

0
0
0

=0 mm/64 mm
=0 mm/64 mm
=0 mm/64 mm

=0
=0
=0

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and Benzil)
The Rf values shows that the compounds (benzoin and benzil) didnt move at all when using
Hexane as the solvent. Hexane is too non-polar, and when the solvents is too low of a polarity,
the component will not move enough, and again separation of the compounds wont occur.
Conclusion:
Thin layer chromatography (TLC) may be used to separate similar compounds. TLC can be used
to help determine the number of components in a mixture, identify the compounds, and the
purity of the compounds. TLC consists of three steps- spotting, development and visualization.
Spotting consists of using a micro-pipette to transfer a small amount of solution at the one end of
the TLC plate. Development consists of placing the bottom of the TLC plate into a beaker
consisting of a development solvent, which then travels up the plate by capillary action. It is
important to use the right solvent when doing this experiment. The last step is visualization, there
are two ways to observe the plate spots (separation of compounds), one is inside the jar with a
few iodine crystals, and another one is under the UV light. In this experiment session, it is more
convenient to just use the UV light, this is because the iodine vapor visualization, because this
takes longer time and some spots are not visible, however by using the UV light, all spots are
visible. The spots are then circled with a pencil and distances are measured. Rf values are
calculated. Rf value is equal to the distance traveled by the substance divided by the distance
traveled by the solvent front. Rf values depends on the polarity of the solvent, the more polar the
compound, the further and have a larger Rf. The less polar, the compounds travel a shorter
distance and have a smaller Rf.

Post-lab questions:
1. Each of the solvents given should effectively separate one of the following mixture by TLC.
Match the appropriate solvent with the mixture that you would expect to separate well with that
solvent. Select your solvent from the following: hexane, methylene chloride, or acetone.
a) 2-phenylethanol and acetophenone
b) Bromobenzene and p-xylene
c) Benzoic acid, 2,4-dinitrobenzoic acid, and 2,4,6-Trinitrobenzoic acid.

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Use methylene chloride for 2-phenylethanol and acetophenone.

Use hexane for Bromobenzene and p-xylene.
Use acetone for Benzoic acid, 2,4-dinitrobenzoic acid, and 2,4,6-Trinitrobenzoic acid.

3. Consider the following errors that could be made when running TLC.
Indicate what should be done to correct the error.
a. A two-component mixture containing 1-octene and 1,4-dimethylbenzene
gave only one spot with an Rf value of 0.95. The solvent used was acetone.
b. A two-component mixture containing a dicarboxylic acid and tricarboxylic
acid gave only one spot with an Rf value of 0.05. The solvent used was
hexane.
c. When a TLC plate was developed, the solvent front ran off the top of the
plate.

a. Acetone polarity (elution solvent) is too high, therefore making the compounds moves on the
same rate. To correct the error, try a new elution solvent mixture which is less polar and the
single spot should separate more efficiently.
b. The Rf value of 0.05 means the components hardly moved at all. Therefore, hexane is too nonpolar and couldnt transfer into the TLC plate. To correct the error, try to use a more polar
solvent.
c. When the TLC plate was developed and the solvent front ran off the top of the top, to correct
this error, use a new TLC plate and run the experiment once again.